Calcium is a critical agent in the activation and deactivation of contraction in the heart. When calcium binds to the myofilaments, contraction is initiated; when calcium comes off the myofilaments, relaxation occurs. Two mechanisms have been proposed for the regulation of relaxation in cardiac tissue: (1) regulation could occur by altering the sensitivity of the myofilaments for calcium, modulating the off rate of calcium from the contractile apparatus, or (2) calcium could be sequestered away from the myofilament, so the calcium necessary for contraction would no longer be available. Beta-adrenergic agonists, such as isoproterenol, both increase the off rate of calcium from the myofilament and increase the rate of calcium sequestration. We monitored these effects by injecting ferret papillary muscles with the photoprotein aequorin, which luminesces in the presence of free calcium. In the presence of isoproterenol, more calcium was needed for a given twitch force, consistent with the hypothesis that the myofilaments were less responsive to calcium, i.e., the off rate of calcium from the myofilaments was increased. The free calcium concentration also fell faster in the presence of isoproterenol, implying a more rapid rate of sequestration. Isoproterenol also accelerated relaxation. The cholinergic agonist acetylcholine was also applied to papillary muscles microinjected with aequorin. For a given level of twitch force, less calcium was required in the presence of acetylcholine than in the absence of drug, suggesting the myofilaments had become more responsive to calcium. However, acetylcholine had no effect on relaxation. When both isoproterenol and acetylcholine were applied to the muscles, the myofilament sensitivity to calcium was not decrease, but relation was accelerated. It is concluded that the modulation of relaxation by isoproterenol and acetylcholine does not occur at the level of the myofilaments.